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At least 1,423 records · Page 79

Nrf2 activation ameliorates cytotoxic effects of arsenic trioxide in acute promyelocytic leukemia cells through increased glutathione levels and arsenic efflux from cells

Carnosic acid (CA), a phenolic diterpene isolated from Rosmarinus officinalis, has been shown to activate nuclear transcription factor E2-related factor 2 (Nrf2), which plays a central role in cytoprotective responses to oxidative and electrophilic stress. Recently, the Nrf2-Kelch ECH associating protein 1 (Keap1) pathway has been associated with cancer drug resistance attributable to modulation of the expression and activation of antioxidant and detoxification enzymes. However, the exact mechanisms by which Nrf2 activation results in chemoresistance are insufficiently understood to date. This study investigated the mechanisms by which the cytotoxic effects of arsenic trioxide (ATO), an anticancer drug, were decreased in acute promyelocytic leukemia cells treated with CA, a typical activator of Nrf2 used to stimulate the Nrf2/Keap1 system. Our findings suggest that arsenic is non-enzymatically incorporated into NB4 cells and forms complexes that are dependent on intracellular glutathione (GSH) concentrations. In addition, the arsenic complexes are recognized as substrates by multidrug resistance proteins and subsequently excreted from the cells. Therefore, Nrf2-associated activation of the GSH biosynthetic pathway, followed by increased levels of intracellular GSH, are key mechanisms underlying accelerated arsenic efflux and attenuation of the cytotoxic effects of ATO. - Highlights: • Nrf2 activation attenuates the effect of arsenic trioxide to acute promyelocytic leukemia cells. • The sensitivity of arsenic trioxide to NB4 cells was dependent on efflux rate of arsenic. • Activation of the GSH biosynthesis is essential in Nrf2-regulated responses for arsenic efflux.

60 APPLIED LIFE SCIENCES↗

Activation of AMPK by berberine induces hepatic lipid accumulation by upregulation of fatty acid translocase CD36 in mice

Emerging evidence has shown that berberine has a protective effect against metabolic syndrome such as obesity and type II diabetes mellitus by activating AMP-activated protein kinase (AMPK). AMPK induces CD36 trafficking to the sarcolemma for fatty acid uptake and oxidation in contracting muscle. However, little is known about the effects of AMPK on CD36 regulation in the liver. We investigated whether AMPK activation by berberine affects CD36 expression and fatty acid uptake in hepatocytes and whether it is linked to hepatic lipid accumulation. Activation of AMPK by berberine or transduction with adenoviral vectors encoding constitutively active AMPK in HepG2 and mouse primary hepatocytes increased the expression and membrane translocation of CD36, resulting in enhanced fatty acid uptake and lipid accumulation as determined by BODIPY-C16 and Nile red fluorescence, respectively. Activation of AMPK by berberine induced the phosphorylation of extracellular signal-regulated kinases 1/2 (ERK1/2) and subsequently induced CCAAT/enhancer-binding protein β (C/EBPβ) binding to the C/EBP-response element in the CD36 promoter in hepatocytes. In addition, hepatic CD36 expression and triglyceride levels were increased in normal diet-fed mice treated with berberine, but completely prevented when hepatic CD36 was silenced with adenovirus containing CD36-specific shRNA. Taken together, prolonged activation of AMPK by berberine increased CD36 expression in hepatocytes, resulting in fatty acid uptake via processes linked to hepatocellular lipid accumulation and fatty liver. - Highlights: • Berberine increases the expression and membrane translocation of CD36 in hepatocytes. • The increase of CD36 results in enhanced fatty acid uptake and lipid accumulation. • Berberine-induced fatty liver is mediated by AMPK-ERK-C/EBPβ pathway. • CD36-specific shRNA inhibited berberine-induced lipid accumulation in liver.

60 APPLIED LIFE SCIENCES↗

Phagolysosome acidification is required for silica and engineered nanoparticle-induced lysosome membrane permeabilization and resultant NLRP3 inflammasome activity

NLRP3 inflammasome activation occurs in response to hazardous particle exposures and is critical for the development of particle-induced lung disease. Mechanisms of Lysosome Membrane Permeabilization (LMP), a central pathway for activation of the NLRP3 inflammasome by inhaled particles, are not fully understood. We demonstrate that the lysosomal vATPases inhibitor Bafilomycin A1 blocked LMP in vitro and ex vivo in primary murine macrophages following exposure to silica, multi-walled carbon nanotubes, and titanium nanobelts. Bafilomycin A1 treatment of particle-exposed macrophages also resulted in decreased active cathepsin L in the cytosol, a surrogate measure for leaked cathepsin B, which was associated with less NLRP3 inflammasome activity. Silica-induced LMP was partially dependent upon lysosomal cathepsins B and L, whereas nanoparticle-induced LMP occurred independent of cathepsin activity. Furthermore, inhibition of lysosomal cathepsin activity with CA-074-Me decreased the release of High Mobility Group Box 1. Together, these data support the notion that lysosome acidification is a prerequisite for particle-induced LMP, and the resultant leak of lysosome cathepsins is a primary regulator of ongoing NLRP3 inflammasome activity and release of HMGB1. - Highlights: • Silica and nanoparticles cause LMP in macrophages in vitro and in vivo. • Phagolysosome acidification is required for particle-induced LMP. • Cathepsin B and L are not required for nanoparticle-induced LMP. • Cathepsin B/L regulate the secretion of HMGB1 with particle exposure.

60 APPLIED LIFE SCIENCES↗

Staphylococcal enterotoxin C2 mutant drives T lymphocyte activation through PI3K/mTOR and NF-ĸB signaling pathways

Staphylococcal enterotoxin C2 (SEC2), a superantigen, causes rapid clonal expansion of lymphocytes and secretion of T cell growth factors, leading to a severe inflammatory response within tissues. Although previous studies have shown that ST-4, a SEC2 mutant with enhanced recognition of Vβ regions of T-cell receptors (TCRVβ), can activate an increased number of T cells and produce more cytokines than SEC2. However, the signaling mechanisms of SEC2/ST-4-mediated immune activation have not been addressed. In this study, we showed that the phosphatidylinositide-3-kinase (PI-3K) inhibitor LY294002, mammalian target of rapamycin (mTOR) inhibitor rapamycin, and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) inhibitor Bay11-7085 could suppress SEC2/ST-4-induced proliferation, CD69/CD25 expression, cell-cycle progression, and IL-2 production in BALB/c mouse splenocytes. In addition, we observed significantly upregulated expression of p70S6K, cyclin E, cyclin D3, and NF-ĸB/p65, but downregulated expression of p27kip during SEC2/ST-4-driven T cells activation. However, SEC2/ST-4-induced changes in cell cycle and PI3K/mTOR signaling were significantly relieved by either LY294002 or rapamycin, and the induction of NF-ĸB/p65 induced was significantly downregulated by Bay11-7085. Moreover, we found that IL-2 secretion was positively associated with p65 expression in a time- and dose-dependent manner. Taken together, our findings demonstrate the involvement of PI3K/mTOR and NF-κB signaling pathways in SEC2/ST-4-induced T cell activation. ST-4 intensifies PI3K/mTOR and NF-ĸB signaling transduction, ultimately leading to enhance T cell activation. These results provide a theoretical mechanism for future immunotherapy using ST-4. - Graphical abstract: SEC2/ST-4 drives T lymphocyte activation through PI3K/mTOR and NF-ĸB signaling pathways - Highlights: • SEC2/ST-4 induces lymphocyte proliferation, CD69, CD25 expression, IL-2 secretion. • SEC2/ST-4 upregulates S6K, cyclin E/D3, p65 expression at mRNA and protein levels. • SEC2/ST-4 downregulates p27kip expression at mRNA and protein levels. • PI3K/mTOR and NF-ĸB inhibitors reverse molecules expression induced by SEC2/ST-4. • ST-4 exhibits higher efficiency than SEC2 in PI3K/mTOR and NF-ĸB pathways.

60 APPLIED LIFE SCIENCES↗

Overexpression of activin-A and -B in malignant mesothelioma – Attenuated Smad3 signaling responses and ERK activation promote cell migration and invasive growth

Activin-A and activin-B, members of the TGF-β superfamily, are regulators of reproductive functions, inflammation and wound healing. These dimeric molecules regulate various cellular activities such as proliferation, migration and suvival. Malignant mesothelioma is an asbestos exposure related tumor affecting mainly pleura and it usually has a dismal prognosis. Here, we demonstrate that both activin-A and -B are abundantly expressed in mesothelioma tumor tissue as well as in cultured primary and established mesothelioma cells. Migratory and invasive mesothelioma cells were also found to have attenuated activation of the Smad2/3 pathway in response to activins. Migration and invasive growth of the cells in three-dimentional matrix was prevented by inhibition of activin activity using a soluble activin receptor 2B (sActR2B-Fc). This was associated with decreased ERK activity. Furthermore, migration and invasive growth was significantly inhibited by blocking ERK phosphorylation. Mesothelioma tumors are locally invasive and our results clearly suggest that acivins have a tumor-promoting function in mesothelioma through increasing expression and switching from canonical Smad3 pathway to non-canonical ERK pathway signaling. Blocking activin activity offers a new therapeutic approach for inhibition of mesothelioma invasive growth. - Highlights: • Activin-A and activin-B are highly expressed in mesothelioma. • Mesothelioma cell migration and invasive growth can be blocked with sActR2B. • Activin induced Smad3 activity is attenuated in invasive mesothelioma cells. • Activins induce ERK activity in mesothelioma cells.

60 APPLIED LIFE SCIENCES↗

Resveratrol upregulates Egr-1 expression and activity involving extracellular signal-regulated protein kinase and ternary complex factors

Many intracellular functions have been attributed to resveratrol, a polyphenolic phytoalexin found in grapes and in other plants. Here, we show that resveratrol induces the expression of the transcription factor Egr-1 in human embryonic kidney cells. Using a chromosomally embedded Egr-1-responsive reporter gene, we show that the Egr-1 activity was significantly elevated in resveratrol-treated cells, indicating that the newly synthesized Egr-1 protein was biologically active. Stimulus-transcription coupling leading to the resveratrol-induced upregulation of Egr-1 expression and activity requires the protein kinases Raf and extracellular signal-regulated protein kinase ERK, while MAP kinase phosphatase-1 functions as a nuclear shut-off device that interrupts the signaling cascade connecting resveratrol stimulation with enhanced Egr-1 expression. On the transcriptional level, Elk-1, a key transcriptional regulator of serum response element-driven gene transcription, connects the intracellular signaling cascade elicited by resveratrol with transcription of the Egr-1 gene. These data were corroborated by the observation that stimulation of the cells with resveratrol increased the transcriptional activation potential of Elk-1. The SRE as well as the GC-rich DNA binding site of Egr-1 function as resveratrol-responsive elements. Thus, resveratrol regulates gene transcription via activation of the stimulus-regulated protein kinases Raf and ERK and the stimulus-responsive transcription factors TCF and Egr-1. - Highlights: • The plant polyphenol resveratrol upregulates Egr-1 expression and activity. • The stimulation of Egr-1 requires the protein kinases ERK and Raf. • Resveratrol treatment upregulates the transcriptional activation potential of Elk-1. • Resveratrol-induced stimulation of Egr-1 requires ternary complex factors. • Two distinct resveratrol-responsive elements were identified.

60 APPLIED LIFE SCIENCES↗

Activation of PPAR alpha by fenofibrate inhibits apoptosis in vascular adventitial fibroblasts partly through SIRT1-mediated deacetylation of FoxO1

Recent studies demonstrated that the ligand-activated transcription factor peroxisome proliferator-activated receptorα (PPARα) acts in association with histone deacetylase sirtuin 1 (SIRT1) in the regulation of metabolism and inflammation involved in cardiovascular diseases. PPARα activation also participates in the modulation of cell apoptosis. Our previous study found that SIRT1 inhibits the apoptosis of vascular adventitial fibroblasts (VAFs). However, whether the role of PPARα in apoptosis of VAFs is mediated by SIRT1 remains unknown. In this study, we aimed to determine the effect of PPARα agonist fenofibrate on cell apoptosis and SIRT1 expression and related mechanisms in ApoE{sup −/−} mice and VAFs in vitro. We found that fenofibrate inhibited cell apoptosis in vascular adventitia and up-regulated SIRT1 expression in aorta of ApoE{sup −/−} mice. Moreover, SIRT1 activator resveratrol (RSV) further enhanced these effects of fenofibrate. In vitro study showed that activation of PPARα by fenofibrate inhibited TNF-α-induced cell apoptosis and cell cycle arrest in VAFs. Meanwhile, fenofibrate up-regulated SIRT1 expression and inhibited SIRT1 translocation from nucleus to cytoplasm in VAFs stimulated with TNF-α. Moreover, the effects of fenofibrate on cell apoptosis and SIRT1 expression in VAFs were reversed by PPARα antagonist GW6471. Importantly, treatment of VAFs with SIRT1 siRNA or pcDNA3.1(+)-SIRT1 showed that the inhibitory effect of fenofibrate on cell apoptosis in VAFs through SIRT1. On the other hand, knockdown of FoxO1 decreased cell apoptosis of VAFs compared with fenofibrate group. Overexpression of FoxO1 increased cell apoptosis of VAFs compared with fenofibrate group. Further study found that fenofibrate decreased the expression of acetylated-FoxO1 in TNF-α-stimulated VAFs, which was abolished by SIRT1 knockdown. Taken together, these findings indicate that activation of PPARα by fenofibrate inhibits cell apoptosis in VAFs partly through the SIRT1-mediated deacetylation of FoxO1. - Highlights: • Fenofibrate inhibits apoptosis and increases SIRT1 expression in vivo and in vitro. • Fenofibrate inhibits cell apoptosis in VAFs through SIRT1. • Fenofibrate inhibits apoptosis through SIRT1-mediated deacetylation of FoxO1. • Fenofibrate inhibits apoptosis and increases SIRT1 expression via PPARα activation.

60 APPLIED LIFE SCIENCES↗

Osteoprotegerin activates osteosarcoma cells that co-express RANK and RANKL

Background: Osteosarcoma (OS) is an aggressive and often fatal cancer that afflicts over 1000 humans and 10,000 dogs per year in the United States. Recent evidence suggests deregulation in the signaling triad, receptor activator of nuclear factor kappa B (RANK), its activating ligand (RANKL), and the RANKL inhibitor, osteoprotegerin (OPG) plays a key role in the pathogenesis of OS. This study investigated the expression of RANK and RANKL in osteosarcoma tumors and cell lines and describes an activating effect of OPG on OS cells in vitro. Results: Canine OS tumors and cell lines co-express mRNA for both RANK and RANKL. Expression of these proteins in OS cell lines was confirmed by Western blot and immunofluorescence microscopy. Expression of the soluble form of RANKL was not detected in media from OS cells. OPG-Fc incubation increased the phosphorylation status of ERK, AKT and the p65 subunit of nuclear factor kappa B (NFκB) and induced NFκB translocation from the cytoplasm to the nucleus in canine OS cells. OPG increased proliferation in both canine and human derived OS cell lines. Conclusion: RANKL is produced by OS tumors and cell lines that also express RANK. This data provides preliminary evidence for a potential autocrine and or paracrine activation pathway in canine OS. An activating effect of exogenous OPG on signal transduction proteins, NFκB and proliferation in OS is described. These data provide new information concerning aberrant signaling in OS and could be important to those considering OPG as a therapeutic agent for osteosarcoma. - Highlights: • Osteosarcoma cells express receptor activator of NFκ-B (RANK) and its ligand RANKL. • Osteoprotegrin (OPG) activates ERK, AKT and NFκB in canine osteosarcoma cells. • Osteoprotegrin incubation causes an increase in total NFκB and phosphorylated NFκB in the nucleus. • Osteoprotegrin increases proliferation in canine and human osteosarcoma cells independent of exogenous RANKL.

60 APPLIED LIFE SCIENCES↗

Electron transport chain inhibitors induce microglia activation through enhancing mitochondrial reactive oxygen species production

Reactive oxygen species (ROS) are believed to be mediators of excessive microglial activation, yet the resources and mechanism are not fully understood. Here we stimulated murine microglial BV-2 cells and primary microglial cells with different inhibitors of electron transport chain (ETC), rotenone, thenoyltrifluoroacetone (TTFA), antimycin A, and NaN{sub 3} to induce mitochondrial ROS production and we observed the role of mitochondrial ROS in microglial activation. Our results showed that ETC inhibitors resulted in significant changes in cell viability, microglial morphology, cell cycle arrest and mitochondrial ROS production in a dose-dependent manner in both primary cultural microglia and BV-2 cell lines. Moreover, ETC inhibitors, especially rotenone and antimycin A stimulated secretion of interleukin 1β (IL-1β), interleukin 6 (IL-6), interleukin 12 (IL-12) and tumor necrosis factor α (TNF-α) by microglia with marked activation of mitogen-activated proteinkinases (MAPKs) and nuclear factor κB (NF-κB), which could be blocked by specific inhibitors of MAPK and NF-κB and mitochondrial antioxidants, Mito-TEMPO. Taken together, our results demonstrated that inhibition of mitochondrial respiratory chain in microglia led to production of mitochondrial ROS and therefore may activate MAPK/NF-kB dependent inflammatory cytokines release in microglia, which indicated that mitochondrial-derived ROS were contributed to microglial activation. - Highlights: • ETC inhibitors induce mitochondrial ROS production in microglia. • ETC inhibitors stimulated secretion of pro-inflammatory cytokines by micriglia. • MAPK/NF-κB pathways maybe related to the microglial activation by ETC inhibitors.

60 APPLIED LIFE SCIENCES↗

NF-κB-dependent transcriptional upregulation of cyclin D1 exerts cytoprotection against hypoxic injury upon EGFR activation

Apoptosis of neural cells is one of the main pathological features in hypoxic/ischemic brain injury. Nuclear factor-κB (NF-κB) might be a potential therapeutic target for hypoxic/ischemic brain injury since NF-κB has been found to be inactivated after hypoxia exposure, yet the underlying molecular mechanisms of NF-κB inactivation are largely unknown. Here we report that epidermal growth factor receptor (EGFR) activation prevents neuron-like PC12 cells apoptosis in response to hypoxia via restoring NF-κB-dependent transcriptional upregulation of cyclin D1. Functionally, EGFR activation by EGF stimulation mitigates hypoxia-induced PC12 cells apoptosis in both dose- and time-dependent manner. Of note, EGFR activation elevates IKKβ phosphorylation, increases IκBα ubiquitination, promotes P65 nuclear translocation and recruitment at cyclin D1 gene promoter as well as upregulates cyclin D1 expression. EGFR activation also abrogates the decrease of IKKβ phosphorylation, reduction of IκBα ubiquitination, blockade of P65 nuclear translocation and recruitment at cyclin D1 gene promoter as well as downregulation of cyclin D1 expression induced by hypoxia. Furthermore, NF-κB-dependent upregulation of cyclin D1 is instrumental for the EGFR-mediated cytoprotection against hypoxic apoptosis. In addition, the dephosphorylation of EGFR induced by either EGF siRNA transfection or anti-HB-EGF neutralization antibody treatment enhances hypoxic cytotoxicity, which are attenuated by EGF administration. Our results highlight the essential role of NF-κB-dependent transcriptional upregulation of cyclin D1 in EGFR-mediated cytoprotective effects under hypoxic preconditioning and support further investigation of EGF in clinical trials of patients with hypoxic/ischemic brain injury. - Highlights: • EGFR activation significantly decreases hypoxia-induced PC12 cells injury. • EGFR activation abrogates the transcriptional repression of cyclin D1 induced by hypoxia in a NF-κB-dependent manner. • NF-κB-dependent cyclin D1 upregulation is required for the EGFR-mediated cytoprotection against hypoxia-induced injury. • Endogenous EGFR activity antagonizes hypoxia-induced PC12 cells injury.

60 APPLIED LIFE SCIENCES↗

Monocyte-lymphocyte fusion induced by the HIV-1 envelope generates functional heterokaryons with an activated monocyte-like phenotype

Enveloped viruses induce cell-cell fusion when infected cells expressing viral envelope proteins interact with target cells, or through the contact of cell-free viral particles with adjoining target cells. CD4{sup +} T lymphocytes and cells from the monocyte-macrophage lineage express receptors for HIV envelope protein. We have previously reported that lymphoid Jurkat T cells expressing the HIV-1 envelope protein (Env) can fuse with THP-1 monocytic cells, forming heterokaryons with a predominantly myeloid phenotype. This study shows that the expression of monocytic markers in heterokaryons is stable, whereas the expression of lymphoid markers is mostly lost. Like THP-1 cells, heterokaryons exhibited FcγR-dependent phagocytic activity and showed an enhanced expression of the activation marker ICAM-1 upon stimulation with PMA. In addition, heterokaryons showed morphological changes compatible with maturation, and high expression of the differentiation marker CD11b in the absence of differentiation-inducing agents. No morphological change nor increase in CD11b expression were observed when an HIV-fusion inhibitor blocked fusion, or when THP-1 cells were cocultured with Jurkat cells expressing a non-fusogenic Env protein, showing that differentiation was not induced merely by cell-cell interaction but required cell-cell fusion. Inhibition of TLR2/TLR4 signaling by a TIRAP inhibitor greatly reduced the expression of CD11b in heterokaryons. Thus, lymphocyte-monocyte heterokaryons induced by HIV-1 Env are stable and functional, and fusion prompts a phenotype characteristic of activated monocytes via intracellular TLR2/TLR4 signaling. - Highlights: • Jurkat T cells expressing the HIV-1 envelope fuse with THP-1 monocytes. • Heterokaryons display a dominant myeloid phenotype and monocyte function. • Heterokaryons exhibit activation features in the absence of activation agents. • Activation is not due to cell-cell interaction but requires cell-cell fusion. • The activated monocyte-like phenotype is mediated by TLR2/TLR4 signaling.

60 APPLIED LIFE SCIENCES↗

High glucose-induced defective thrombospondin-1 release from astrocytes via TLR9 activation contributes to the synaptic protein loss

Highlights: • High glucose (30 mM) impaired TSP-1 secretion from astrocytes, resulting in neuronal synaptic proteins loss. • High glucose (30 mM) induced TLR9 activation and ROS generation in astrocytes. • Defective TSP-1 secretion from astrocytes was mediated by TLR9-ROS signaling. Diabetes, characterized by chronic hyperglycemia, is known to induce synaptic degeneration in the brain, thereby resulting in cognitive dysfunction. Thrombospondin-1(TSP-1), the secreted protein produced by astrocytes, plays a crucial role in promoting synapse formation. Toll-like receptor 9 (TLR9) has been widely known to initiate the innate immune response. We recently reported TLR9 activation in neurons results in tau hyperphosphorylation induced by HG in vitro. Its activation has been also considered to mediate oxidative stress and astrocytic dysfunction under pathological circumstance. However, whether astrocytic TSP-1 alteration plays a role in synaptic protein loss under high glucose condition and whether TLR9 activation is involved in this process have not been reported. In this study, we found that primary mouse astrocytes incubated in high glucose (30 mM) induced a significant decreased TSP-1 secretion and increased intracellular contents of TSP-1 without affecting transcription level. Addition of conditioned medium from high glucose (30 mM) treated astrocytes to the primary neurons exhibited reduced synaptic proteins expression, which was attenuated by treatment with exogenous rTSP-1. In addition, we demonstrated that TLR9 activation along with reactive oxygen species (ROS) generation in astrocytes was induced by high glucose (30 mM). Furthermore, we explored the relationship between TLR9 activation and TSP-1 production. Both TLR9 deficiency and the antioxidant N-acetyl-L-cysteine treatment improved altered intra- and extracellular TSP-1 levels under high glucose condition. Together, our findings suggest that high glucose (30 mM) impairs TSP-1 secretion from astrocytes, which depends on astrocytic dysfunction associated with TLR9 activation mediated ROS signaling, ultimately contributing to the synaptic proteins loss.

60 APPLIED LIFE SCIENCES↗

Artemin regulates CXCR4 expression to induce migration and invasion in pancreatic cancer cells through activation of NF-κB signaling

Highlights: • Artemin regulates CXCR4 expression to induce migration and invasion in pancreatic cancer cells through activation of NF-κB signaling. • Artemin activates Akt and ERK, which cooperatively mediates activation of NF-κB and subsequent CXCR4 up-regulation. • Artemin induces an increase in migration and invasion potential in pancreatic cancer cell by regulating Akt and ERK1/2/NF-κB/CXCR4 signaling. Pancreatic ductal adenocarcinoma (PDAC) is the most lethal human malignant tumor because of the early onset of local invasion and distant metastasis. Perineural invasion is a prominent characteristic of pancreatic adenocarcinoma, which is a multifactorial process that involves various signaling molecules from different signaling pathways. The glial cell line-derived neurotrophic factor family of ligands was reported to be involved in perineural invasion in pancreatic cancer. Artemin is one member of the glial cell line-derived neurotrophic factor family of ligands. Although Artemin has previously been demonstrated to promote invasiveness of pancreatic cancer, the mechanisms remain poorly understood. In this study, we performed an analysis to determine the effects of Artemin on modulating tumor cell metastatic potential and invasion activity and explored its mechanisms in pancreatic cancer. We indicated that Artemin and CXCR4 were overexpressed in cancer tissues and widely expressed in pancreatic cancer cell lines. We observed that activation of ERK1/2 and Akt in Artemin-treated cells led to enhanced nuclear accumulation of NF-κB, which then induced CXCR4 expression. Through regulation of the expression of CXCR4, Artemin functionally promoted the migration and invasion in pancreatic cancer cells. The present study indicated that Artemin induced CXCR4 expression by activating Akt and ERK 1/2/NF-κB signaling, thereby modulating tumor cell metastatic potential and invasion activity in pancreatic cancer by regulating SDF-1α/CXCR4 axis. Artemin might be an effective and potent therapeutic target for pancreatic cancer metastasis, especially in perineural invasion.

60 APPLIED LIFE SCIENCES↗

Vangl2 is essential for myocardial remodeling activated by Wnt/JNK signaling

Highlights: • Wnt5a triggered activation of JNK in time-dependent manner. • Activation of Wnt/JNK signaling stimulates myocardial remodeling. • Vangl2 knockdown partly reversed myocardial remodeling. The Wnt/JNK pathway, responsible for tissue polarity in cardiogenesis in vertebrates, has been shown to play numerous roles during differentiation and development of cardiac myocytes. Van Gogh-like-2 (Vangl2) is a core component that regulates the induction of polarized cellular and tissue morphology during animal development. However, little is known about Wnt/JNK signaling pathway in the process of myocardial remodeling. In present study, we found that activation of Wnt/JNK signaling by Wnt5a stimulates enlargement of cardiomyocyte surface area. The hypertrophic features were inhibited in Vangl2 depleted cells. Meanwhile, Wnt/JNK activation induced cytoskeleton rearrangement but failed to activate these effects in cells lacking Vangl2. Moreover, Wnt/JNK activation significantly increased the cell apoptosis by mediating the mitochondrial permeability transition pore (mPTP) dysfunction, whereas knockdown of Vangl2 partly reversed these effects. These results suggest that activation of Wnt/JNK signaling stimulates myocardial remodeling (cell morphological changes, apoptosis and mitochondrial dysfunction), in which Vangl2 may play an essential role.

60 APPLIED LIFE SCIENCES↗

Glycyrrhizin ameliorates inflammatory pain by inhibiting microglial activation-mediated inflammatory response via blockage of the HMGB1-TLR4-NF-kB pathway

Highlights: • Glycyrrhizin (Gly) represses LPS-induced microglial activation without cell toxicity. • Glycyrrhizin dose-dependently inhibits LPS-triggered inflammatory response. • Elevation of HMGB1 reverses glycyrrhizin-inhibited inflammatory response in microglia. • HMGB1-mediated TLR4-NF-κB activation account for gly-restrained inflammation. • Gly alleviates CFA-induced pain hypersensitivity, inflammation, microglia activation. Chronic inflammatory pain is a severe clinical problem that greatly affects patients’ quality of life and causes huge economic burden. Microglia-mediated neuroinflammation exerts critical roles in the pathogenic progression of inflammatory pain. Recent evidence corroborates the anti-inflammatory and neuroprotective efficacy of glycyrrhizin; however, its function in inflammatory pain remains poorly elucidated. In the present study, glycyrrhizin suppressed LPS-induced activation of microglial cell BV2 by inhibiting NO production and expression of microglial marker IBA-1. Intriguingly, LPS-induced high expression and generation of inflammatory cytokines (i.e., IL-6, TNF-α and IL-1β) was notably reversed by glycyrrhizin pre-treatment. Mechanistic analysis confirmed that high expression of high-mobility group box 1 (HMGB1) in LPS-activated microglia was inhibited following glycyrrhizin. More importantly, restoring HMGB1 expression by recombinant adenovirus vector of Ad-HMGB1 counteracted glycyrrhizin-restrained inflammatory response in microglia upon LPS stimulation. Furthermore, glycyrrhizin dampened the activation of subsequent TLR4-NF-κB pathway in LPS-stimulated microglia, which was abrogated by HMGB1 elevation. Furthermore, blocking this pathway by si-TLR4 transfection reversed the effects of HMGB1 overexpression on the inhibitor roles of glycyrrhizin in microglia-triggered inflammation. Additionally, glycyrrhizin administration also alleviated CFA-evoked mechanical allodynia and thermal hyperalgesia in inflammatory pain model of mice, concomitant with suppression in inflammatory response and microglial activation. Simultaneously, elevation of HMGB1, TLR4 and p65-NF-κB protein expression induced by CFA injection was also abrogated after glycyrrhizin. Accordingly, this study reveal that glycyrrhizin may act as a promising therapeutic avenue for the treatment of inflammatory pain.

60 APPLIED LIFE SCIENCES↗

Exogenous hydrogen sulfide protects from endothelial cell damage, platelet activation, and neutrophils extracellular traps formation in hyperhomocysteinemia rats

Highlights: • NaHS protects from ECs damage, platelet activation and NETs formation. • NaHS regulates platelets activation concern the phosphor-p38 MAPK pathway. • The inhibition of NaHS on platelets activation which prevents the NETs formation. Hydrogen sulfide (H{sub 2}S) prevents endothelial cells damage and P-selectin of platelets promotes neutrophils extracellular traps (NETs) formation. However, how sodium hydrosulfide (NaHS), a donor that produces H{sub 2}S regulates the activation of platelets and whether H{sub 2}S inhibits the formation of neutrophils extracellular traps in hyperhomocysteinemia rats have not been previously investigated. The morphological and ultrastructural alterations of endothelial cells (ECs) and platelets were tested by transmission electron microscopy. The expressions of P-selectin of platelets were determined by flow cytometry. Additionally, the cellular ROS and the H{sub 2}S level were detected by DCFH-DA staining and H{sub 2}S probe, the expressions of Bax and Bcl-2 in arteries and cultured ECs from rat thoracic aortas and the phosphor-p38 mitogen-activated protein kinase (MAPK), CSE and CBS of platelets were measured by western blotting. The NETs formations, the concentration of DNA in serum and supernatant of cultured neutrophils stimulated with platelet-rich plasma (PRP) were tested by Sytox Green and PicoGreen commercial Kits. The vascular ECs damaged, the expression of P-selectin of platelets and NETs formation increased; the concentration of DNA in serum and supernatant of cultured neutrophils stimulated with PRP also increased; the expression of Bax increased while Bcl-2 decreased in arteries, the phosphor-p38 MAPK of platelets increased while CSE and CBS have no statistically significant changes in the HHcy group compared to the control group. In the cultured ECs, the ROS level increased while the H{sub 2}S level decreased after 48 and 72 h treatment by HHcy; the expression of Bcl-2 decreased while Bax increased after 72 h treatment by HHcy. NaHS significantly inhibited the ECs injured, cellular ROS production, platelet activation and NETs formation, reversed the expressions of Bax, Bcl-2, phosphor-p38 MAPK, P-selectin and the increased concentration of DNA in serum and supernatant of cultured neutrophils which caused by high homocysteine. Our results demonstrate that the donor of H{sub 2}S inhibits the platelets activation and NETs formation, which concerts the protection of ECs in hyperhomocysteinemia.

60 APPLIED LIFE SCIENCES↗

Lipid accumulation-induced hepatocyte senescence regulates the activation of hepatic stellate cells through the Nrf2-antioxidant response element pathway

Highlights: • Oxidative stress caused by lipid deposition causes hepatocyte senescence. • Free fatty acid-induced senescent hepatocytes promote hepatic stellate cell activation and development of hepatic fibrosis. • Senescent hepatocytes regulate hepatic fibrosis through the Nrf2-antioxidant response element pathway. Non-alcoholic fatty liver disease (NAFLD) has become the most prevalent chronic liver disease globally. Elderly individuals are at a higher risk of developing NAFLD with severe clinical outcomes. Although NAFLD is closely related to liver aging, the role of hepatocyte senescence in the progression of NAFLD, especially in the development of fibrosis, is still unclear. The early stage of NAFLD is mainly characterized by lipid accumulation in hepatocytes, which could lead to severe oxidative stress, causing cellular senescence. In the present study, hepatocytes cultured in the presence of free fatty acids to induce lipid deposition were used as a hepatocyte senescence model in vitro. Senescent hepatocytes significantly increased the activation of co-cultured primary hepatic stellate cells (HSCs) and the expression of pro-fibrosis molecules. Moreover, the antioxidant regulator nuclear factor erythroid 2-related factor 2 (Nrf2) that was upregulated in senescent hepatocytes was found to be related to the activation of co-cultured HSCs. The Nrf2 agonist sulforaphane, which upregulated the transcriptional activity of the Nrf2-antioxidant response element (ARE) pathway, remarkably inhibited hepatocyte senescence and its activation effect on HSCs. However, the liver tissue obtained from non-alcoholic steatohepatitis (NASH) mice with Nrf2 knockdown showed decreased antioxidation and significant liver senescence and fibrosis. In conclusion, this study confirmed that lipid accumulation induces hepatocyte senescence, which leads to HSC activation and development of hepatic fibrosis. Increasing the activity of the Nrf2-ARE antioxidant pathway in senescent hepatocytes elicited the opposite effect, suggesting that targeting Nrf2 may prevent or delay the progression of aging-related liver fibrosis in NASH.

60 APPLIED LIFE SCIENCES↗

Atomic-scale Imaging of PGM-free Catalyst Active Sites by 30 keV 4D-STEM

Platinum group metal (PGM)-free catalysts have attracted a large amount of attention due to their potential for enabling low-cost, commercially viable hydrogen fuel cells and electrolyzers [1]. Degradation of cell performance remains a significant challenge, however, currently limiting implementation of devices that utilize PGM-free materials [2]. Controlling degradation in these materials requires a better understanding of the atomic structure of active sites, thought to be FeN4 structures within a graphitic carbon lattice, which would enable more accurate prediction of the associated degradation pathways. The exact structural arrangement of active sites in these materials is still debated since a range of structures have been computationally predicted and methods to directly validate these models are still needed [1,3-5].While scanning transmission electron microscopy (STEM) has provided initial glimpses into the nature of the proposed active sites, detailed evaluation of the structure of these sites is challenging since they involve defects, edges, and nitrogen dopants in the graphitic lattice, which are susceptible to beam damage [6,7]. Conventional STEM imaging and spectroscopy methods exacerbate this problem with dose-inefficiency or nonideal contrast characteristics for imaging light and heavy elements simultaneously. A method that minimizes damage while generating dose-efficient, easily-interpretable contrast for both light and heavy elements is therefore needed to facilitate imaging of sensitive active site atomic structure.Here, we demonstrate direct atomic-scale imaging of PGM-free catalyst active sites by low-voltage four-dimensional (4D)-STEM. To accomplish this, we pair a 30 keV aberration-corrected probe, which minimizes knock-on damage, with a fast pixelated detector that has optimal performance at low beam energies [8]. We show how this setup enables relatively simple center-of-mass (CoM) techniques [9,10] to produce images with an increased signal-to-noise ratio (SNR) and light-element contrast over conventional imaging modes, allowing the entire active site structure to be imaged at the atomic scale. Moreover, we show how electron ptychography [11,12] enables images with further improved characteristics to be obtained, for example by minimizing residual aberrations, which provides a more accurate structural representation. The increased understanding that these low-voltage 4D-STEM techniques will provide about the atomic structure of PGM-free active sites and their associated degradation pathways will facilitate rational design of next-generation materials, promoting development of low-cost hydrogen fuel cells, electrolyzers, and other energy conversion devices [13]. References:[1] U Martinez et al., Adv. Mater. 31 (2019), p. 1806545.[2] Y Shao et al., Adv. Mater. 31 (2019), p. 1807615.[3] J Kneebone et al., J. Phys. Chem. C 121, (2017), p.16283.[4] T Mineva et al., ACS Catal. 9 (2019), p. 9359.[5] J Li et al., Nat. Catal. 4 (2021), p. 10.[6] T Susi et al., ACS Nano 6 (2012), p. 8837.[7] P Zelenay and DJ Myers, US Department of Energy Hydrogen and Fuel Cells Program 2017 Annual Merit Review and Peer Evaluation Meeting, Washington, DC (2017).[8] H Ryll et al., J. Inst. 11 (2016), p. P04006.[9] K Müller et al., Nat. Commun. 5 (2014), p. 5653.[10] I Lazic et al., Ultramicroscopy 160 (2016), p. 265.[11] H Yang et al., Nat. Commun. 7 (2016), p. 12532.[12] Y Jiang et al., Nature 559 (2018), p. 343. [13] Research sponsored by the Hydrogen and Fuel Cell Technologies Office, Office of Energy Efficiency and Renewable Energy, US Department of Energy (DOE). Research was conducted at the Center for Nanophase Materials Sciences, which is a DOE Office of Science User Facility.

Zachman, Michael↗